Cytoplasmic antigens for detection of candida

ABSTRACT

The present invention relates to a method and a means of diagnosing  Candida  infection. In particular the present invention relates to a method of diagnosing  Candida  infection by measuring the levels of antibody to  Candida  cytoplasmic antigen present in a biological sample taken from a subject at risk of, or suspected to be suffering from a  Candida  infection.

FIELD OF THE INVENTION

The present invention relates to a method and a means of diagnosingCandida infection. In particular the present invention relates to amethod of diagnosing Candida infection which is both sensitive andrapid.

BACKGROUND OF THE INVENTION

Candida is the most commonly identified causative agent of oral orvaginal thrush. However, over the last few decades Candida has emergedas a significant cause of life-threatening infections in hospitalpatients. Ironically the increasing incidence of these “invasive” or“systemic” Candida infections has been advances in modern medicine.Patients that are now surviving major injuries, surgery, cancers andorgan transplants are vulnerable to life-threatening Candida infections.In the United States, Candida is now the fourth most common cause ofblood infections in hospitals.

The major problem with systemic Candida infections is that there are fewdefinitive clinical signs or symptoms. Treatment is largely based onsuspicion rather than a definitive diagnosis. Even with the availabilityof anti-fungal drugs such as fluconazole a high mortality rate (30 to70%) is associated with systemic Candida infections. The high rate ofmortality is largely due to the rapid onset of infection and a rapidlyfatal outcome. Without an accurate diagnosis the infection often goesunnoticed until it is too late to effectively treat. This has led to acomment by clinicians that Candida infections are usually diagnosed atautopsy. Accordingly, there is a need for a rapid diagnostic assay thatis capable of early diagnosis of Candida infection so that appropriatetreatment may be instituted thereby reducing the mortality rate.

The main difficulty in the diagnosis of Candida infections is that beinga commensal, mere isolation of Candida from body surfaces, or orifices,is not diagnostic of an infection. Culture of Candida from blood or deeptissue is still the main method of diagnosis of systemic Candidainfections. However, it can take several days for a culture to becomepositive, by then it may be too late to effectively treat the infection.Also, false positives may occur due to contamination from superficialbody sites. Of more importance, is the observation that in up to fiftypercent of autopsy proven cases of systemic candidiasis, blood cultureswere negative and therefore of no diagnostic value.

Nuclear magnetic resonance (NMR) and radioisotope scanning have beenused to detect Candida infections in tissues and organs. However, thosemethods are not useful for early diagnosis.

Recently analysis of the Candida metabolite arabinitol was proposed as adiagnostic tool. However, as arabinitol is produced by the human body,further clinical studies have cast doubt on its value.

The polymerase chain reaction (PCR) has also been used in the diagnosisof invasive Candida infections. However, PCR has not established itselfas a useful diagnostic method for Candida for the same reasons asoutlined above ie Candida is a ubiquitously present microorganism andfalse positives, due to superficial contamination, are prevalent.

Immunoassays are the established procedures for the diagnosis of manytypes of infectious diseases. Immunoassays have the advantage that theyare rapid and have a standardised assay format. Immunoassays can bedesigned to either detect Candida antigens, or host antibodies reactiveagainst Candida antigens. Several immunoassays are commerciallyavailable for the detection of Candida antigens in sera or other bodyfluids. However, these assays lack either sensitivity or specificity orboth.

Immunoassays have been developed based on the detection ofimmunodominant Candida antigens. Candida mannan is a highly immunogeniccell wall antigen. However, as Candida is a commensal, most individualshave antibody to Candida mannan, so its usefulness in the diagnosis ofsystemic infection is limited. The applicant has now surprisingly foundthat a more discriminatory assay for Candida than previously used is thedetection of cytoplasmic antigen. The advantage of this diagnostic assayis that antibody to this cytoplasmic antigen is only produced inresponse to an actual infection. The applicant has further demonstratedthat the use of a combination of cytoplasmic antigen with other antigensis very predicative of Candida infection.

Accordingly, the present invention overcomes or at least alleviates theproblems normally associated with diagnosing Candida infection.

SUMMARY OF THE INVENTION

In its most general aspect, the invention disclosed herein provides asimple and rapid method for diagnosis of Candida infection. The methodof diagnosis of Candida infection may be used to screen large numbers ofsamples for possible infection.

Accordingly, in one aspect, the invention provides a method ofdiagnosing Candida infection, comprising the steps of:

a). obtaining a biological sample from a subject at risk of, orsuspected to be suffering from, Candida infection, and

b). measuring the levels of antibody to Candida cytoplasmic antigenpresent in the biological sample.

Antibody levels may be measured using known techniques of immunologyincluding enzyme-linked immunoassay (ELISA or EIA), biligand binding(sandwich technique), fluorometric assay, chemiluminescent assay,immunochromatography, radialimmunodiffusion or radioimmunoassay (RIA).ELISA, immunochromatography or chemiluminescent assay methods areparticularly preferred, since these are quick, sensitive, and specific,and are readily automated for large-scale use. These methods alsoprovide quantitative determinations.

The diagnostic method utilises antigens expressed by Candida, especiallycytoplasmic antigen. The antigens isolated from Candida as disclosedherein may, in certain embodiments of the diagnostic method of thepresent invention, be immobilised on an inert surface, embedded in agel, or may be conjugated to a molecule which imparts colour,fluorescence or radioactivity to the antigen.

In a second aspect, the invention provides a method for assessing theprognosis of Candida infection, comprising the steps of measuring thelevels of antibody to Candida cytoplasmic antigen in a biologicalsample.

Persons skilled in the art will appreciate that the techniques disclosedherein may be used on any type of biological sample. Preferable thebiological sample is selected from the group consisting of bone marrow,plasma, spinal fluid, lymph fluid, the external sections of the skinfrom respiratory, intestinal, and genitourinary tracts, tears, saliva,milk, blood; both whole blood and sera, blood cells, tumours and organs.Most preferably the biological sample is sera.

Biological samples that may be analysed by the method of the presentinvention can also be obtained via swabs, shunts or the like. Thebiological samples may be analysed directly, or may be treated prior totesting by, for example, concentration or pH adjustment.

In a third aspect, the present invention further provides a method ofdetecting the presence or absence of a Candida antibody comprising thesteps of:

a). exposing a biological sample, which may include a Candida antibody,to an isolated cytoplasmic Candida antigen; and

b). detecting the reaction between antibody and antigen.

In an especially preferred embodiment of the present invention thediagnostic assay further utilises other Candida antigens in combinationwith the cytoplasmic antigen. In particular the cell wall antigen(including mannose) and/or purified immunodominant antigen (enolase) areutilised.

Accordingly, in a forth aspect of the present invention there isprovided a method of diagnosing Candida infection, comprising the stepsof:

a). obtaining a biological sample from a subject at risk of, orsuspected to be suffering from, Candida infection, and

b). measuring the levels of antibody present in the biological sample toCandida cytoplasmic antigen in combination with measuring the levels ofantibody to either cell wall antigen or immunodominant antigen (enolase)or both.

The reagents and means of diagnosis of the present invention may also beembodied in a kit for use in a diagnostics laboratory or may be adaptedand automated for analysing large numbers of samples at a centralreceiving centre.

Accordingly, in a fifth aspect the invention provides a kit when usedfor detecting the presence or absence of a Candida antibody in abiological sample, comprising:

a). a biological sample collection device;

b). a cytoplasmic Candida antigen; and

c). means for detecting reaction between the antibody and antigen in thesample.

Suitable buffering agents and ionic salts may also be included in thekit.

In a sixth aspect the invention provides a method of preparing acytoplasmic antigen comprising the step of removing lipoproteins bychloroform extraction.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a coomassie blue stained SDS-PAGE with major protein bandsof the Candida cytoplasmic antigen fraction observed at 55 kDa, 35 to 45kDa region, 30 kDa and 20 kDa.

FIG. 2 shows a single coomassie blue band of 48 kDa corresponding to theexpected size of the enolase antigen.

FIG. 3 shows a coomassie blue stained gel of the clarified cell wallantigen preparation. A broad smear of stain can be seen ranging in sizefrom 90 kDa to 200 kDa

FIG. 4 shows a number of sera screened against the Candida cytoplasmicantigen preparation.

FIG. 5 shows antibody reactivity to the three Candidaantigens—cytoplasmic, cell wall and immunodominant antigens, usingnegative control sera.

FIG. 6 shows antibody reactivity to the three Candidaantigens—cytoplasmic, cell wall and immunodominant antigens, using serafrom patients with superficial candidiasis.

FIG. 7 shows antibody reactivity to the three Candidaantigens—cytoplasmic, cell wall and immunodominant antigens, using serafrom patients with systemic candidiasis.

FIG. 8 shows the error bar of the Applicant antigen test values in thedifferent blood culture patients (95% CI).

FIG. 9 shows an error plot of the mean Candida antibody values measuredby the Applicant antigen test in both the blood culture positive andnegative groups of patients (95% confidence interval).

FIG. 10 shows an error bar graph of the Applicant antigen test data forinvasive candidiasis and healthy controls.

FIG. 11 shows an immunoblot in which panel A shows the C. albicanscytoplasmic (enolase) antigen disclosed in Buckley et al. compared withpanel B, C. albicans antigen disclosed in the present application.

ABBREVIATIONS USED

EDTA Ethylenediaminetetraacetic acidEIA Enzyme immunoassayELISA Enzyme-linked immunosorbent assay

RIA Radioimmunoassay

BSA Bovine serum albuminDMSO Dimethyl sulfoxideβ-Me β-mercaptoethanolTMB 3,3′,5,5′-tetramethyl-benzidine

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The practice of the present invention employs, unless otherwiseindicated, conventional molecular biology, cellular biology, andimmunoassay techniques within the skill of the art. Such techniques arewell known to the skilled worker, and are explained fully in theliterature. See, e.g., Harlow and Lane, “Antibodies: A LaboratoryManual” (1988); Maniatis, Fritsch & Sambrook, “Molecular Cloning: ALaboratory Manual” (1982); “Animal Cell Culture” (R. I. Freshney, ed.,1986); “Immobilised Cells and Enzymes” (IRL Press, 1986); B. Perbal, “APractical Guide to Molecular Cloning” (1984); Sambrook, et al.,“Molecular Cloning: a Laboratory Manual” (1989) and Ausubel, F. et al.,1989-1999, “Current Protocols in Molecular Biology” (Green Publishing,New York).

In describing the present invention, the following terminology is usedin accordance with the definitions set out below.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a individual, including but not limited to bonemarrow, plasma, serum, spinal fluid, lymph fluid, the external sectionsof the skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood; both whole blood and anti-coagulated whole blood,blood cells, tumours, organs, and also includes samples of in vivo cellculture constituents, including but not limited to conditioned mediumresulting from the growth of cells in cell culture medium, putativelyCandida infected cells, recombinant cells, and cell components.

“Human tissue” is an aggregate of human cells which may constitute asolid mass. This term also encompasses a suspension of human cells, suchas blood cells, or a human cell line.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

It will be clearly understood that, although a number of prior artpublications are referred to herein, this reference does not constitutean admission that any of these documents forms part of the commongeneral knowledge in the art, in Australia or in any other country.

Persons skilled in the art will appreciate that any number of differentimmunoassays may be used in the present invention. For example, theCandida antigens disclosed herein may be used in antibody captureassays, antigen capture assays, wherein the antigen/antibody complexforms a “special” class of antigen or two-antibody sandwich assays.

Techniques Used for Antigen Preparation

The term “Candida antigen” as used here means any one of the threeseparate types of Candida antigen utilised in the present invention,namely, cell wall antigen (including mannose), total cytoplasmic antigen(mannose depleted) or purified immunodominant antigen (enolase). Use ofthe term “Candida antigens” means that all three antigens were involvedor could be utilised. A number of techniques may be used to prepare theCandida antigens including biochemical extraction, columnchromatography, Gel fractionation, gene cloning, differentialprecipitation, filtration, dialysis or centrifugation; however, thepreferred techniques are those disclosed herein. Briefly, thesetechniques involve either mechanical, chemical or enzymatic lysis ofCandida cells, followed by separation of insoluble cell walls fromsoluble cytoplasmic fraction by centrifugation, filtration and dialysis.Chemical treatment of cell wall fraction to release cell wall antigensfollowed by centrifugation and dialysis. Filtration and organicextraction of soluble cytoplasmic cell extract. Separation ofmannoproteins by ConA affinity chromatography. Purification of theimmunodominant enolase antigen from the soluble cytoplasmic extract byanion and cation affinity chromatography. It will be appreciated bythose skilled in the art that other techniques, or modifications orvariations of the above techniques, may be adopted without adverselyaffecting the spirit of the present invention.

Techniques Used for Antibody Preparation and Labelling

Antiserum to the Candida antigens disclosed herein may be produced in ahost animal such as rabbit or sheep. The serum fraction containing theantibody may be isolated by standard techniques. This antiserum may beemployed in several of the embodiments of the invention hereinafter setforth, or a more sensitive and specific antibody might be obtained byfurther purification of the serum by electrophoresis, high-speedcentrifugation or the like. Ultimately, large quantities of highlyspecific monoclonal antibody may be produced by means of thehybrid-myeloma techniques by methods known to those skilled in the art.

Certain embodiments of the present invention employ antibody to theCandida antigens immobilised on cellulose, agarose, sephadex or glassbeads or other similar inert surfaces such as metal, plastic or ceramicwhich do not interfere with subsequent reaction. Adsorption, Br—CNactivation or other techniques known in the art may be employed toimmobilise the antibody.

Other embodiments of the present invention employ the antibody to theCandida antigens conjugated to a chromophoric (highly coloured)molecule, an enzochromic (an enzyme which produces colour upon additionof reagents) molecule, fluorochromic (fluorescent) molecule or aluminogenic (luminescent) molecule.

The conjugate of antibody with enzyme is made using techniques known inthe prior art. (For references, see Avrameas, S. and Uriel, J., inComptes Rendus Hebdomadaires des Seances de l'Academie des Sciences,vol. 262, p. 2543, (1966); Nakane, P. K. and Pierce, G. B., in Journalof Histochemistry and Cytochemistry, vol. 14, p. 929, (1966); Nakane, P.K., in methods in Enzymology, vol. 37, p. 133, (1975)).

Chromophoric molecules that may be used are 2,3-dinitrobenzene (DNB)salts, dinitrophenol (DNP) and methyl and butyl orange. Other suitablechromophoric agents are well known in the art. Enzochromic moleculesthat may be conjugated with the antibody are enzymes that give colourwith appropriate reagents. Examples are alkaline phosphatase (ALP) whichdevelops colour with nitrophenyl phosphate (NPP), glucose oxidase withglucose, and D-galactopyranoside. These and other examples are wellknown in the art. Examples of fluorogenic agents are2,4-dinitrofluorobenzene and “pipsyl” derivatives. Luminogenic moleculesmay be conjugated to antibodies by the method of Branchini, et al.(Biochem. Biophys. Res. Commun. 97, 334 [1980]). The term “chromophoric”hereinafter is intended to include “enzochromic” “fluorochromic” and“luminogenic” molecules as well.

Certain embodiments of the invention also utilise Candida antibodytagged with a radioactive element. I¹²⁵ conjugated by means of thechloramine-T procedure is a common example, but other methods known inthe art may also be employed.

Techniques Used for Antigen Immobilisation and Labelling

Antigen molecules may be immobilised on a solid carrier by a variety ofmethods known in the art, including covalent coupling, directadsorption, physical entrapment and attachment to a protein-coatedsurface. For references describing the methodology, see Silman, I. H.and Katchalski, E. in Annual Review of Biochemistry, Vol. 35, p. 873(1966); Melrose, G. J. H., in Review of Pure and Applied Chemistry, Vol.21, p. 83, (1971); and Cuatrecasas, P. and Anfinsen, C. B., in Methodsin Enzymology, Vol. 22, (1971).

Lai et al. (German OS No. 2,539,657, U.S. Pat. No. 4,066,512) disclosesa method of attachment to a protein-coated surface. In this method, theinternal and external surfaces of a microporous membrane are firstcoated with a water-insoluble protein such as zein, collagen,fibrinogen, keratin, glutelin, polyisoleucine, polytryptophan,polyphenylalanine, polytyrosine, or copolymers of leucine with p-aminophenylalanine. Such a coating renders the membrane capable ofimmobilising a wide variety of biologically active proteins includingenzymes, antigens, and antibodies. A microporous structure is defined asone having more than 50% of its total volume in the form of poresranging in size from 25 nanometres to 25 micrometers, preferably from 25nanometres to 14 micrometers. A pore size range from 25 nanometres to 5micrometers is employed in most applications herein. Uncoatedmicroporous membranes have as much as 70 to 75% of their volume as porespace. The pores permit liquid flow through the membrane. After beingcoated by zein, for example, the pore space is reduced 5 to 10% with theresult that the structure retains its essential properties of having ahigh proportion of its volume as pore space and permitting liquid flowthrough the pores. The structure has a large surface area in contactwith any solution contained within the pores.

Such a coated membrane, having immobilised antigen or antibody, providesa compact, easy to manipulate carrier for the immobilised antigen orantibody. Its integral structure permits removal of bound from unboundcomponents by simple mechanical means.

Non-specific binding may be minimised by interposing a second stageimmobilisation step, in which an immunochemically neutral protein isimmobilised to the filter. Immobilisation therefore occurs in two stagesaccording to a preferred embodiment of the invention: a first stage inwhich the desired immunochemical component is immobilised, and a secondstage, following the completion of the first, in which animmunochemically neutral protein such as fetal calf serum or bovinegamma globulin is next immobilised. The term immunochemically neutral isdefined in terms of the specific components of the assay. Any protein,which does not combine immunochemically with a component of the assay orwith one of the reagents, is considered immunochemically neutral, eventhough such protein might be immunochemically reactive in anothersystem.

Where the substance to be detected is an antibody, the immunochemicallyreactive moiety of the conjugate must be an antibody capable of bindingimmunochemically with the antibody to be tested. Such antibodies may beobtained by immunising an animal with the antibody or immunoglobulinfraction of serum from the animal in which the antibody to be testedoriginated. For example, where the antibody to be tested is a humanantibody, a goat antibody against human antibody is obtained from theserum of a goat immunised against human immunoglobulin (antibody). Theenzyme moiety may be any enzyme capable of catalysing a reaction whichcan be detected by any method known to those skilled in the art, andwhich retains its activity after conjugation with antibody. Horseradishperoxidase is preferred because of its convenience and suitability to awide range of applications. It is well known that the enzyme catalysesthe oxidations of a variety of organic compounds in the presence ofhydrogen peroxide. Many such organic substrates are chromogenic, ie.undergo a colour change upon oxidation.

It has been found in the present invention that the purity of the enzymepreparation used in the formation of conjugate has an effect on thedegree of non-specific binding. The greater the purity of the enzymepreparation, the less the non-specific binding. In part, the reductionis made possible because, the total amount of conjugate protein requiredis reduced as the specific activity of the enzyme is increased. Theopportunity for non-specific binding is therefore reduced as well. Inthe preferred embodiment, the use of a highly purified peroxidasepreparation has been found to significantly reduce the amount of colourreaction observed in control samples as compared with known positives.

Techniques Used for Candida Antibody Detection Antibody CaptureTechnique

A Candida antigen prepared by the techniques disclosed herein isimmobilised, preferably on an inert surface such as PVC, paper or asimilar bibulous mat. The immobilised Candida antigen is then put intocontact with a sample suspected of containing Candida antibody. In thecase of aqueous samples such as blood or urine, the solution is bufferedand ionic salts may be present at optimum concentration for Candidaantibody-Candida antigen interaction. TRIS or borate buffered phosphateat pH 7.5 to 9.0 and ionic strength about 0.010 to 0.5, for example, aresuitable buffering agents and ionic salts. The inert surface withCandida antigen or Candida antigen-Candida antibody complex thereon isnext put into contact with antibody to Candida antigen conjugated to achromophormic molecule. Preferably the Candida antigen is in solutionbuffered at pH from about 7.5 to 9.0 and ionic concentration equivalentto about 0.01M to about 0.1M NaCl. After careful rinsing under water orwith suitable surfactants such as Tween 20 to remove excess colouredantibody, the inert surface is inspected for colour, fluorescence orluminescence directly or after addition of colour-developing agents.Colour on the inert surface indicates interaction between immobilisedCandida antigen-Candida antibody complex in solution. A control may berun for colour comparison.

This technique may be adapted to clinical use by employing Candidaantigens tagged with radioactive elements and observing either depletionof activity in solution or uptake on solid support of radioactivity.This embodiment is highly sensitive and rapid and suitable for largenumbers of samples.

Enzyme-Linked Immunoassay-ELISA

A solution comprising Candida antibody conjugated to enzyme which formscolour with developing reagents and buffer and ionic salts suitable forreaction between Candida antigen and the Candida antibody is put intocontact and allowed to react with Candida antigen immobilised,preferably, on an inert surface such as PVC, paper strip or glass bead.The amount of enzochromic conjugated Candida antibody is sufficient tosaturate about 50% of the reactive sites on the immobilized antibody.The inert surface with antibody-Candida antigen enzyme complex is putinto contact with buffered sample suspected of containing Candida, saidsample having an unknown amount of Candida antibody. The colour of theresultant immobilised antibody-Candida antigen-enzyme complex on thestrip after colour developing reagents are added is observed incomparison to a control strip which has not been treated with samplecontaining Candida antibody. Dilution in colour on inert surface treatedwith sample means presence of Candida antibody in the unknown sample.

This method may be adapted for clinical use by contacting samples andimmobilised enzyme, preferably in tubes which may be centrifuged andwatching developing colour spectrophotometrically. This embodiment isvery sensitive and rapid.

Radialimmunodiffusion-Precipitin Reaction

One of the Candida antigens is suspended in a softened gelatinous mediumsuch as agar or agarose along with buffers and salts to maintain pHbetween about 6.0 to 9.0 and ionic strength between about 0.01M to 0.5Mfor optimal antigen-antibody interaction. The suspending medium of U.S.Pat. No. 4,259,207 is a suitable example. The mixture is spread out toharden on a test plate or, preferably, poured into a disc-shapedcontainer such as an Octolony plate. A small amount of sample is placedon the solidified gel, preferably in a centre well and the plate or discis allowed to stand preferably covered for a period of hours. Diffusionof sample into the surrounding area occurs during this period. If theCandida antibody is present, it reacts with the embedded Candida antigenand causes an opaque area in a radial pattern about the point ofapplication of sample. A control can be run for comparison. Calibrationof an amount of Candida antibody in the sample, if desired, can beobtained by controlling temperature, time and size of sample andcomparing the resultant size of radial area with one of knownconcentration.

Radioimmunoassay

A Candida antigen of the present invention is immobilised on an inertsurface such as glass beads in a separation column. A portion of Candidaantigen is conjugated to a radioactive element, preferably 1125 andallowed to react with the immobilised Candida antigen in an amountsufficient to, saturate 50% of the binding sites. The immobilisedCandida antigen-enzyme complex is put into contact with a samplesuspected of containing Candida antibody, the sample being bufferedbetween pH 6-9 and containing total ionic salts about 0.05 to 0.5M foroptimal reaction conditions for formation of Candida antigen-antibodycomplex. The Candida antibody is eluted from the antigen and the eluantis measured for radioactivity. Loss of activity compared to a controlindicates Candida antibody in the sample.

Haemagglutination

Candida antibody may be assayed through standard haemagglutinationtechniques with Candida antigen to antibody used as sensitising agent.

It is to be understood that methods described hereinabove for assay ofCandida antibody employing coloured reagents have been presented mostspecifically for application where neither trained personnel norsophisticated instruments are available. These methods, however, may beadapted for use in a clinical setting where large numbers of samples areto be assayed by substituting radioactive elements for chromogenicconjugated molecules.

It is also to be understood that the term “colour” is not to beinterpreted as being limited to the narrow visible range of theelectromagnetic spectrum, but is meant to include wavelengths which maybe measured by standard spectrophotographic instruments such asspectrophotometers and absorption and emission colourimeters in both theuv and the ir range.

Although it is contemplated that the methods of the present inventionare to be applied to biological fluids themselves, the sensitivity andspecificity of the method can be improved by culture of the fluidspreferably on medium selective for Candida prior to testing.

Sensitivity may also be improved by preliminary treatment of biologicalsamples with lysing agents such as isotonic solution, sound, or lysozymeto release Candida antibody into the extracellular environment. U.S.Pat. No. 4,166,765, for example, discloses suitable lysing proceduresfor biological samples containing bacteria. Any lysing agent may beemployed which does not interfere with subsequent enzyme activity.

Assays Embodied in Kit Form

The diagnostic method and means of the present invention may be embodiedin the form of a kit for use by individuals for self-diagnosis ofCandida in the privacy of their homes.

The kit comprises a means for sample collection, the Candida antigen toCandida antibody and a means for detecting reaction between sample andCandida antigen.

In embodiments adapted for clinical use, electrophoretic separationtechniques such as isoelectric focusing or zone electrophoresis whichare based on differences of both size and charge distribution betweenproducts and reactants may likewise be used to separate products fromreactants. Products separated electrophoretically may be detected bycharacteristic locations compared to standards or may be identified bycolour or immunochemically. Resinous beads of charged surfaces may alsobe used to separate products and reactants.

The means for detecting reaction in the case of immunoassay in apreferred embodiment of the invention is a gelatinous medium in whichthe Candida antigen to antibody is suspended. The gelatinous medium isin a transparent glass or plastic container and comprises buffer andionic salts for optimal conditions for formation of the Candidaantigen-antibody complex. Reaction is noted as a transparent arearadiating from the central point at which the sample is applied.

The means for detecting reaction in another preferred embodimentcomprising immunoassay is the Candida antigen to Candida antibodyconjugated to a chromophore in a sealed, sterile packet along withbuffer and ionic salts. For assay, the contents of the packet arediluted with water in a marked tube supplied in the kit. Included alsoin this embodiment is the antigen to Candida antibody immobilised on aninert surface. For assay, the inert surface with immobilised Candidaantigen is put into contact with sample and then with the solution ofchromophore-conjugated Candida anti-IgA antibody, protein A or proteinG. The inert surface is inspected for colour, which indicates Candida.

In a particularly preferred embodiment, the kit of the present inventionis provided in the form of an immunochromatographic test strip device.There are many patents that cover a number of technologies, formats,reagents and materials that may be of great value in the development andproduction of immunochromatographic test strip devices. For example,U.S. Pat. No. 5,075,078, International Patent Application No.WO95/16207, U.S. Pat. No. 5,654,162 and European Patent No. 0810436A1.The assay methods used with the devices disclosed in these patents areessentially the same. A ligand specific for the analyte (normally, butnot necessarily an antibody [Ab]) is immobilised to a membrane such asnitrocellulose. The detector reagent, typically an antibody coupled tolatex or colloidal metal, is deposited (but remains unbound) into theconjugate pad. When sample (urine, plasma, whole blood, etc.) is addedto the sample pad, it rapidly wets through to the conjugate pad and thedetector reagent is solubilised. The detector reagent begins to movewith the sample flow front up the membrane strip. Analyte that ispresent in the sample will be bound by the antibody that is coupled tothe detector reagent. As the sample passes over the zone to which thecapture reagent has been immobilised, the analyte detector reagentcomplex is trapped. Colour develops in proportion to the amount ofanalyte present in the sample.

In the present case, while the above principles are the same, ratherthan detecting analyte per se, the immunochromatographic test stripdevice would detect antibody. In such situations, it would be theantigen(s) disclosed herein which would immobilised onto membranes,sample pads, reagent pads and other porous media rather than antibody.There are a wealth of information regarding the development of suchdevices including methods of binding antigen/antibodies tonitrocellulose and the like and detecting such bound material. See forexample, Towbin et al. 1979, Proc. Natl. Acad. Sci. USA 76:4350, theentirety of which is included herein by reference.

Although the invention has been described with reference to presentlypreferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Moreover, the following examples are offered by way ofillustration only and are not intended to limit the invention in anymanner. All patent and literature references cited herein are expresslyincorporated.

Example 1 Preparation of Candida Antigen

The following three types of Candida antigen were prepared:

1). Cell wall antigen (including mannose);

2). Total cytoplasmic antigen (mannose depleted); and

3). Purified immunodominant antigen (enolase).

A clinical isolate of the Candida albicans, was obtained from a patientwith vaginal thrush. The identity of the Candida species was confirmedwith the use of an API 20C Auxonagram strip (API System S.A., France).The C. albicans isolate was designated KEMH5.

200 ml YEPD culture medium (1% yeast extract, 2% peptone, 2% D-glucose)was inoculated with the isolate as a starter culture and incubated for24 h at 30° C. with aeration. The starter culture was then used toinoculate a 10 L YEPD culture incubated under similar conditions in a 23L Bio-Flo Fermenter IV System (New Brunswick Scientific, Edison, N.J.).

The Candida culture was harvested from the Bio-Flo fermenter system andseparated from culture medium with the use of a Pellicon filtrationcassette (Millipore, USA). Concentrated cells were separated fromresidual medium by centrifugation in 500 ml centrifuge flasks for 15 minat 1,660×g and 4° C. The supernatant was discarded and the pelletedcells were resuspended in protein extraction buffer (20 mM bis-Tris, pH6.5). The yeast cells were then centrifuged as described previously,resuspended and pooled for further processing.

Candida cells were ruptured mechanically with the use of a Dynomill®(WAB, Switzerland). Milling was continued until 99% cell disruption wasobtained. The soluble Candida cell extracts were collected and dispensedinto 50 ml centrifuge tubes. The extracts were centrifuged for 12 h at8,517×g and 4° C. to precipitate insoluble cell walls. The supernatantscontaining the soluble cytoplasmic antigen fraction were recovered andpassed through a 0.45 μm filter membrane.

The filtrates were then extracted with an equal volume of chilledchloroform. Following centrifugation at 4° C. for 15 min at 1,036×g theupper aqueous phase was aspirated and transferred to a dialysis tube.The soluble cytoplasmic protein fractions were dialysed in columnbinding buffer (20 mM Tris/HCl, pH 7.4, 0.5M NaCl, 1 mM MnCl₂.4H₂0, 1 mMCaCl₂) for 12 h in preparation for chromatography.

The soluble cytoplasmic antigen fraction was depleted of contaminatingsoluble cell wall mannoprotein by Con A-Sepharose chromatography. Thedialysed cytoplasmic antigen fraction was filtered through a 0.45 μmfilter. 50 ml of the dialysed extract was applied onto a Con A-Sepharosecolumn (2.6×12.5 cm) equilibrated in binding buffer at a flow rate of 4ml/min. The unbound flow-through fraction (non-glycosylated proteins)was collected. Bound mannoproteins were eluted with 0.5M α-methylmannoside in binding buffer. This step was performed before the next runand to clean the column before storage.

The soluble cytoplasmic antigen fraction was dialysed overnight against20 mM Tris.Cl, pH7.4. An estimate of the quantity of protein in solutionwas performed using the Bio-Rad® (Bradford) microassay procedure inaccordance with the manufacturers instructions. A portion of thecytoplasmic antigen extract was analysed by SDS-PAGE.

As shown in FIG. 1 there was a number of major protein bands observedwhich varyed in size from approx 20 kDa up to approx 60 kDa in size. Themajor staining bands being at 55 kDa, four bands in the 35 to 45 kDaregion, 30 kDa and 20 kDa. This was in stark contrast to the largenumber of Coomassie blue staining bands in the original crude lysateprior to organic extraction and Con A-Sepharose chromatography.

Purification of the enolase antigen was conducted in the same fashion asthe soluble Candida cytoplasmic antigen except that it was not subjectedto Con A-Sepharose chromatography. Instead, following dialysis andfiltering through a 0.20 μm syringe filter (cellulose acetate), thefiltered extracts were applied to a Pharmacia Biotech XK 50/20chromatography column packed with Pharmacia Biotech Source 15Qquaternary ammonium anion exchanger (Pharmacia LKB, Uppsala, Sweden).The column was equilibrated prior to chromatography with column bindingbuffer ‘A’ (20 mM bis-Tris, pH 6.5). Anion exchange chromatography ofthe crude extracts was controlled and recorded using the Bio-Rad® Econo®system (Bio-Rad Laboratories, USA). Bound protein was eluted from thecolumn with a salt gradient of buffer ‘B’ (1M NaCl in buffer ‘A’, pH6.5). The recovered fractionated proteins were analysed by an enzymeactivity assay.

The active enzyme enolase hydrolyses D(+)₂-phosphoglyceric acid (PGA) tophosphoenolpyruvate (PEP). The production of PEP can be monitored byspectrophotometry at 240 nm. 20 μl of protein solution was combined with1 ml of enolase substrate solution (50 mM Tris-HCl pH 7.4, 2.7 mMmagnesium acetate, 1.0 mM EDTA, 1.2 mM D(+)₂-phosphoglyceric acid) in aquartz cuvette and the change of absorbance recorded at 1 min intervals.The specific activity was defined as the conversion of 1 μmol of PGA toPEP per min per mg protein. An estimate of the quantity of protein insolution was performed using the Bio-Rad® (Bradford) microassayprocedure.

Eluate fractions containing enolase activity were selected and dialysedfor 12 h at 25° C. in hpH₂O. The dialysed fractions were recovered andfiltered through a 0.20 μm syringe filter. The filtrate was concentratedten-fold by evaporation under vacuum for 5 h. The concentrated sampleswere dialysed with binding buffer ‘A’ (10 mM sodium acetate, pH 4.7)immediately prior to application to a Pharmacia Biotech Mono S HR10/10chromatography column packed with methyl sulphonate cation exchanger(Pharmacia LKB, Uppsala, Sweden). Cation exchange chromatography wasperformed using the Bio-Rad® Biologic system. Bound protein fractionswere eluted from the column with a salt gradient of buffer ‘B’ (1M NaClin buffer ‘A’, pH 4.7). Fractions containing enolase activity wereidentified by the enzyme activity assay described above.

FIG. 2 shows a single Coomassie blue band of 48 kDa corresponding to theexpected size of the enolase antigen. The identification of the 48 kDaantigen as the glycolytic enzyme enolase was confirmed by an enolaseactivity assay.

Purification of the cell wall antigen was conducted as follows: theprecipitated insoluble cell walls were collected followingcentrifugation as described above. The cell walls were washed with hpH₂Othen collected by centrifugation at 6,000 rpm. This step was repeatedthree times or until the supernatant was no longer cloudy. This ensuredany residual soluble cytoplasmic antigen was removed from the cell wallpreparation. The washed cell wall pellet was then resuspend in 10 mMPhosphate buffer pH7.4 containing 1% v/v β-Me and incubated for 30 minat 37° C. in a shaker to solubilise the cell wall antigens. The samplewas then centrifuged for 5 min at 8,000 rpm and the pellet was thendiscarded. The supernatant was transferred into a fresh tube andrecentrifuged (5 min at 8,000 rpm). The supernatant containing thesolubilised cell wall antigen was then dialysed in hpH₂O for 48 h at 4°C. (four changes of water), or until no odour was detected. Followingdialysis the sample was centrifuged three times 5 min at 8,000 rpm toremove any residual particular matter.

Following clarification the cell wall antigen preparation was analysedby SDS-PAGE. The resulting Coomassie blue stained gel is presented inFIG. 3. A broad smear of stain is seen ranging in size from 90 kDa to200 kDa. The lack of discrete protein bands is typical of mannoproteins,where differences in the number of mannose groups added to the proteinbase results in a variety of molecular weights.

Example 2 Enzyme Linked Immunosorbent Assays (ELISAS)

A serum panel was collected from 1998 to 2000 from various patients withCandida infections. Negative control (Control) sera (n=20) were obtainedfrom the Red Cross Blood Bank, Perth, Australia and was obtained fromhealthy males in the 19 to 25 year age group. Sera (n=13) from patientswith recurrent vulvo vaginal candidiasis (VVC) were obtained from KingEdward Memorial Hospital, Perth, Australia. Sera (n=108) from patientswith oral candidiasis were obtained from Clinipath Ltd and the UWADental School, Perth, Australia. Sera (n=39) from patients (n=28) withsystemic candidiasis were obtained from Princess Margaret Hospital,Perth, Australia and Prince of Wales Hospital, Sydney, Australia.

In the case of patients with oral and vaginal Candida infection,confirmation of infection was made by physical examination and byculture of Candida organisms from the relevant body site. In the case ofpatients with systemic infection, confirmation of infection was throughpositive blood culture or biopsy. In all cases the immune status of thepatient was unknown.

Sera from patients with either superficial or systemic candidiasis werescreened by ELISA using trays coated with the Candida cytoplasmicantigen. The protein content of each antigen preparation was determinedusing a commercial assay (BioRad) with BSA as a standard. A series ofELISAs were performed to determine the optimal coating concentration foreach antigen (data not shown). The optimal coating concentration beingthat which gave the greatest discrimination between a positive and anegative control serum. For each antigen the optimum coatingconcentration was determined to be 2 μg/ml.

A 96 well C8 strip microtitre plate (Greiner GmbH, Germany), was coatedwith either Candida cell wall antigen, cytoplasmic antigen, or purifiedenolase antigen as prepared in Example 1. 50 μl of a 2.0 μg/ml solutionof the antigen was diluted in coating buffer (0.1M NaHCO₃, pH 9.3) andadded to individual wells. The plates were incubated for 12 h at 4° C.then equilibrated to ambient temperature. After equilibrating the platesto ambient temperature, coating solution was decanted and the platetapped dried. Plates were inverted on paper towel to drain.Alternatively excess coating solution was aspirated by the automatedplate washer (Dynatech Laboratories, Chantilly Va., USA). It wasimportant not to wash the plate at this stage.

A volume of 300 μl of blocking solution (PBS pH 7.3, 2% (w/v) BSA (ICN,Australia), 0.01% (w/v) Tween 20), was applied to each well andincubated at 25° C. for 90 min. Blocking solution was decanted and theplate tapped dried. Plates were inverted on paper towel to drain andtapped dried for a second time. At this stage plates were either usedimmediately, or dried for storage. Plates to be dried were placedinverted in a sealable container such as a plastic food container with anumber of silica gel desiccant sachets for 48 h. The inclusion ofapproximately 20 small desiccant sachets was adequate for the drying of6 coated ELISA micro-well trays. Dried plates were sealed intoheat-sealed packets with a single desiccant sachet and labelled. Plateswere stored at 4° C. until required. Packets containing plates wereequilibrated to ambient temperature before opening.

Human test sera diluted 1/100 in blocking solution was dispensed intowells in 50 μl aliquot's and incubated at 37° C. for 30 min. The primaryantibody solution was aspirated and wells were washed six times inPBS-Tween 20. The plates were inverted on paper towels and allowed todrain for 10 min. The plates were then tapped dried.

A volume of 100 μl of a horseradish peroxidase anti-human IgG conjugatediluted 1/10,000 in blocking solution was dispensed to each well.Secondary antibody solution was incubated at 37° C. for 30 min. Thesecondary antibody solution was aspirated and wells were washed sixtimes in PBS-Tween 20. Plates were inverted on paper towel to drain for10 min and then tapped dried. Plates were inverted on paper towel for asecond time and allowed to drain for 5 min. Plates were then tappeddried. Particular care was employed to ensure that all traces ofsecondary conjugate solution was removed as residual conjugate wasestablished as the major factor responsible for disparity of results(Dynatech Laboratories Inc, USA).

A volume of 100 μl of TMB liquid substrate solution was dispensed intoeach well and developed at 25° C. for 10 min. The reaction wasterminated with the addition of 100 μl of 1M phosphoric acid or 1MH₂SO₄. The absorbance values for each well were measured at 450 nm,reference 620 nm with a MRX automated plate reader.

Each immunoassay was performed in triplicate and the mean value ofabsorbance was used. The absorbances are shown as a Scatter diagram inFIG. 4. Three groups of patients with Candida infections were analysed.The first group were patients with systemic candidiasis (Systemics), thesecond group had oral candidiasis (Oral) and the third group hadvulvovaginal candidiasis (VVC). Blood bank sera (Control) from males inthe 19 to 25 year age group, who were at low risk of having anundetected or subclinical Candida infection were used as a control. Thecut-off absorbance (OD₄₅₀=0.22) was the mean value of the negativecontrol sera. From these data the cytoplasmic antigen ELISA had asensitivity of 89% and a specificity of 95%. This is higher than thatreported for other Candida serological tests (Zoller et al., 1991. J.Clin. Micro. 29:1860-1867).

To further increase the sensitivity of the Candida ELISA multipleantigens were used. These were the cell wall, cytoplasmic and nativeenolase (described above).

The use of multiple antigens increased the sensitivity of the CandidaELISA. It also provided greater discrimination between superficial andsystemic infection. Six negative control sera (serum obtained fromhealthy males in the 19 to 25 year age group) were used in ELISAs withmicrotitre tray wells individually coated with the three Candidaantigens. For each serum the antibody titre to each of the threeantigens was below that of the cut-off line (FIG. 5). This line is thecut off value assigned based on a comparison of the average antibodytitres of sera from control patients versus those of candidiasispatients. The value plotted on the y-axis of the graph is the ratio ofthe cut-off absorbance divided into the absorbance of the test serum.

Serum obtained from 6 patients with superficial candidiasis was thenreacted in the ELISA. Again the absorbance value of each serum wasdivided by the absorbance of the cut-off (FIG. 6). The characteristicantibody response of the sera from patients with superficial candidiasiswas a high titre against the cell wall antigen preparation (1.5 to 2times the cut-off value). The antibody reactivity to the completecytoplasmic antigen preparation was positive in most cases (1 to 1.5times the cut-off). In contrast the antibody titre to the enolaseantigen was below or equal to that of the cut-off. There is acorrelation between the antibody titre to the internal Candida antigens(cytoplasmic and enolase) and the severity of the superficial infection(data not shown). However, the severity of the infection in the sixpatients analysed was not known.

Six sera taken from patients with systemic candidiasis (confirmed bypositive blood culture) were analysed by ELISA. The results arepresented in FIG. 7. In the case of the patients with systemiccandidiasis the antibody response to the cell wall antigen preparationwas positive (1.5 to 2 times the cut-off value). Also, the antibodytitres to the internal Candida antigens (cytoplasmic and enolase) werealso positive (1.5 to 2.5 times cut-off value).

CONCLUSIONS

The Candida mannan depleted cytoplasmic antigen preparation disclosedherein can be used to identify patients with Candida infections. Thesensitivity and specificity using an ELISA with microtitre trays coatedwith this antigen is greater than that obtained by other Candidadiagnostic tests. Further, the ELISA assay format disclosed herein iseasier to perform, more robust and more rapid than formats used in otheravailable Candida diagnostic assays. The ELISA format also has theadvantage that it is quantifiable. This enables the patient to bemonitored over a period of time and changes in the titre of the antibodyresponse to the Candida antigens recorded. The ability of the test tomonitor overtime the antibody titre to Candida antigens has a prognosticvalue in terms of measuring the patient's response to antifungal drugsand in the overall survival prospects of the patient. Another advantageof the cytoplasmic antigen preparation is that the method developed toproduce the antigen is simpler and more rapid than other availableprocedures (eg. compare with that of Zoller et al., 1991, supra).

Example 3 Clinical Evaluation in France

Clinical evaluation of the triple antigen test kit as described inExamples 1 and 2 was undertaken in the Department of Parasitology andMedical Mycology at the University of Grenoble Faculty of Medicine,Grenoble, France using stored sera.

Sera from two groups of patients were analysed: those that were bloodculture positive and those that were blood culture negative. Whenpossible, sera were taken before, at the time of and after the first dayof positive blood culture to be tested. The blood culture negative groupwas divided into 3 subgroups: Patients that were colonised with Candidaand were serology positive, patients that were colonised with Candidaand were serology negative, and patients that were not colonised withCandida and serology negative. The sera were obtained from patientshospitalised between 1998 and 2000.

The triple antigen ELIZA test (“the Applicant antigen test”) wasperformed according to Example 2. The cut-off calibrator sera wasobtained by pooling sera taken from males in 19 to 25 age group who hadno history of Candida infections.

Table 1 shows that the Applicant antigen test was positive in 15 out of19 patients who had a positive blood culture.

TABLE 1 APPLICANT TRIPLE ANTIGEN TEST AS USED IN THE FRENCH STUDY Dateof Applicant serum Triple Ag Applicant relative Abs/Cut Triple AgSerology- to first Applicant off Score immuno- Patient Patient SerumCandida +ve Triple Ag (0.46) (0.46 fluorescence IEP IEP Ag Group ID IDspecies culture Abs ratio cut-off) IFI Pasteur FSK emie Candidemia AMIC1 C. g −4 1.597 3.5 +++ +++ AMI C2 +3 1.519 3.3 +++ ++ +++ +++ BRIG C3C. a −2 0.385 0.8 − − BRIG C4 +10 0.325 0.7 − − COE C5 C. t −13 0.4060.9 − − COE C6 +1 0.734 1.6 + +++ COE C7 +29 0.632 1.4 + − COH C8 C. g−14 0.597 1.3 + − COH C9 +9 0.661 1.4 + − COH C10 +65 0.391 0.9 − − COMC11 C. g +1 1.806 3.9 +++ + +++ ++ COM C12 +19 1.862 4.0 +++ ++ ++ +++CON C13 C. g −27 0.732 1.6 + − + CON C14 +1 0.5 1.1 (+) − ++ CON C15 +80.367 0.8 − − ++ DA SI C17 C. a +2 1.805 3.9 +++ + ++++ ++ DA SI C18 +701.277 2.8 ++ ++ ++ (+) FER C19 C. a −35 0.693 1.5 + − FER C20 +2 0.3680.8 − − − FER C21 +16 0.229 0.5 − − FON C22 C. a −46 0.51 1.1 (+) − FONC23 +3 1.899 4.1 +++ +++ FON C24 +27 1.854 4.0 +++ +++ HAM C25 C. a +11.083 2.4 ++ + + + HAM C26 +31 1.168 2.5 ++ + ++ (+) HEN C27 C. t +20.324 0.7 − − HEN C28 +7 0.646 1.4 + − HEN C29 +40 0.432 0.9 − − KHA C30C. a −13 0.332 0.7 − − KHA C31 +2 1.553 3.4 +++ + KHA C32 +27 1.393 3.0+++ + LON C33 C. a −2 0.341 0.7 − − LON C34 +6 0.447 1.0 (+) − LON C35+61 0.35 0.8 − − MAN C36 C. a −28 0.505 1.1 (+) − MAN C37 +5 0.288 0.6 −− MAN C38 +72 0.199 0.4 − − NI C39 C. t & +3 0.223 0.5 − − NI C40 C. k+9 0.368 0.8 − ++ + + PAS C41 C. a +5 0.865 1.9 + + ++ + PASe C42 +321.279 2.8 ++ + ++ ++ + PIL C43 C. p −2 0.495 1.1 (+) − PIL C44 +51 0.8311.8 + + RAM C45 C. t +5 1.414 3.1 +++ ++ RAM C46 +23 1.114 2.4 ++ + NOIC47 ? 0 0.611 1.3 + +++ Hospital ABE D21 No No info. 0.748 1.6 + +patients info. that are FRE D22 No No info. 0.454 1.0 − − colonisedinfo. but have BEN D23 C. a urine 0.331 0.7 − − negative BER D24 C. tmouth/fae 0.463 1.0 − − Candida BOM D25 C. a broncal/ 1.046 2.3 ++ ++serology fae CAP D26 No No info. 0.658 1.4 + − info. CAR D27 C. atrachea 0.933 2.0 ++ + CHE D28 C. t urine 1.376 3.0 +++ − FER D29 C. aurine/faeces 0.363 0.8 − − GIN D30 C. g urine/faeces 0.663 1.4 + − PERD31 C. a thorax 0.378 0.8 − − drain Hospital BEN D32 C. spp urine 0.4691.0 − − patients BON D33 — 0.44 1.0 − − that are CIA D34 C. a urine 0.922.0 ++ ++ non- DAVID D35 — 0.651 1.4 + − colonised CAR and have PEL D36— 0.752 1.6 + − negative DI M D37 — 0.489 1.1 (+) − Candida FEU D38 —0.633 1.4 + − serology FOG D39 C. a thorax 1.095 2.4 ++ + drain MOR D400.38 0.8 − − GO D41 0.677 1.5 + − Hospital ALL D42 C. spp urine 1.1772.6 ++ + patients BAR D43 C. g, septic 1.375 3.0 +++ +++ that are C. a,shock colonised C. t and have BOE D44 C. g urine/mouth 1.096 2.4 ++ ++positive BUI D45 No No info. 1.125 2.4 ++ ++ Candida info. serology COLD46 No No info. 1.062 2.3 ++ +++ info. DAG D47 No No info. 0.705 1.5 + −info. BE D48 C. a & mouth/trachea 1.123 2.4 ++ +++ C. t GEN D49 No Noinfo. 1.426 3.1 +++ ++++ info. GEN D50 No No info. 1.489 3.2 +++ ++++info. LEC D51 No No info. 1.668 3.6 +++ +++++ info. LECr D52 No No info.1.62 3.5 +++ +++++ info. Legend: Candida Culture Sp. Applicant Ab IFIIEP Pasteur IEP FSK Ag-emie C. a = C. albicans <10 = − <20 = − 1 arc = +1 arc = + ½ dil = + C. g = C. glabrata 10-20 = + 20 = + 2 arc = ++ 2 arc= ++ ¼ dil = ++ C. k = C. kefir 20-30 = ++ 40 = ++ 3 arc = +++ 3 arc =+++ C. p = C. parapsilosis 30-40 = +++ 80 = +++ 4 arc = ++++ 4 arc =++++ C. t = C. tropicalis 160 = ++++ 320 = +++++Of the 12 patients who had sera taken before or on the day of the firstpositive blood culture, 8 gave a positive (or low positive) result. Whencompared with other serology tests used by the French group 12 out of 19patients were positive by the immunofluorescence (IFI) serology test.All but one of these was positive using the Applicant antigen test. Onepatient was also positive by the Applicant antigen test, but negative byIFI. All of the 5 patients that tested positive by IEP Pasteur, IEP FSKor Ag-emie serology tests were also positive by the Applicant antigentest.

It is possible that some of the patients that were negative by both theApplicant and the IFI test may have had a transient candidemia due tocentral line contaminations.

Six of 11 patients that were know to be colonised, but had negativeserology were positive by the Applicant antigen test. Two of thepositive patients were also positive by IFI. Of the 10 non-colonisedhospital patients with negative serology six were positive by theApplicant antigen test, two of these positive patients were alsopositive by IFI. All nine patients that were colonised patients withpositive serology were positive by the Applicant antigen test. Thesedata compared to 8 out of 9 patients that were positive by IFI. The onlyIFI negative sample was a low positive by the Applicant antigen test.

The statistical analysis of these data is presented in FIG. 8 and Table2.

TABLE 2 Mean 95% Confidence Category (Units) Interval CandidemiaPatients 21.79^(a,b) 16.25-27.33 Colonised + negative 14.55^(a,c) 9.67-19.42 Serology Non-colonised + 14.2^(b,d) 10.68-17.72 negativeserology Colonised + positive 27.27^(c,d) 23.12-31.43 serology ^(a)p =0.71 ^(b)p = 0.58 ^(c)&^(d)p < 0.01In the candidemia patients with positive blood culture to Candida, themean of their Candida antibody levels detected by Applicant antigen testwas 21.79 (16.25-27.33 95% CI). Using the Independent Samples T-test,the p value was 0.71 between the candidemia group and the colonisedgroup with negative serology to Candida. The p value was 0.58 betweenthe means of the candidemia group and the non-colonised group that wasnegative for Candida serology.

For the negative blood culture patients, the patients in the groups thatwere negative for Candida serology had generally lower Candida antibodylevels detected by the Applicant antigen test. The mean antibody levelswere 14.55 units in the colonised group (9.67-19.42, 95% CI) and 14.2units in the non-colonised group (10.68-17.72, 95% CI). These levelswere significantly lowered (p<0.01) from the mean antibody levels in thegroup of patients that had positive Candida colonisation culture resultsand positive Candida serology, mean 27.27 (23.12-31.43, 95% CI). Theseare clearly seen in the error bars in FIG. 8.

Overall, there was a good correlation with the Applicant antigen testand other tests used. There was also a good correlation with the titreof antibody detected by the Applicant antigen test and the level ofpositiveness of the other tests ie., a patient that had a high positiveresult with the Applicant antigen test also had a similar result withthe other tests used (eg, patients AMI, COM, DA SI, FON, PAS and RAM).Similarly patients that were negative or low positive with the Applicantantigen test were also negative or weak positive by the other tests (eg,patients BRIG, FER, HEN, LON and MAN). It was noted that some of theblood culture negative patients were positive by the Applicant antigentest, which demonstrated the great sensitivity of the Applicant antigentest.

Example 4 Clinical Evaluation in Spain

A similar clinical evaluation to that undertaken in Example 3 wasconducted by Professors Guillermo Quindós, MD, PhD, Maria DoloresMoragues, PhD, and Jose Pontón, PhD, Department of Immunology,Microbiology and Parasitology at the Faculty of Medicine, University ofPai's Vasco, Bilbao, Spain.

The retrospective study sera were obtained from 11 patients (Table3—Patients 1.1 to 1.32) with invasive candidiasis as defined by positiveblood culture or by histology and positive tissue biopsy. The “bloodculture negative” group consisted of sera from 12 patients (Table4—Patients 2.2 to 2.53) selected on the basis of the patients have arisk of invasive candidiasis but having negative blood cultures. Between3 and 5 sera were tested per patient. For patients withmicrobiologically proven candidiasis the sera were taken before, at thetime of and after the positive blood culture. For the blood culturenegative group the sera were taken at various times duringhospitalisation. As well as sera from hospital patients, sera from threehealthy blood donors were also tested (Table 5). Also a group of freshsera were prospectively tested from 5 patients, two with positiveCandida blood cultures and 3 without (Table 5).

TABLE 3 Patients With Positive Blood Culture Day Triple PlateliaPlatelia Spanish Spanish Candida species Patient extraction Ag Ag AbAnti-B Anti-GT & Outcome 1.01 −21 + + + ++++ + C. albicans 1.01−11 + + + ++++ ++ 1.01 0 + + + ++++ ++ 1.01 7 + ++ ++ +++ +++ 1.01 13+++ + ++ +++ +++ 1.11 −1 +++ ++ +++ ++++ ++ C. glabrata 1.11 8 +++ +++++ ++++ +++ 1.11 21 +++ + + +++ +++ 1.11 29 +++ + +++ ++++ ++++ Exitusd45 1.17 −11 +++ ++ +++ ++++ − C. albicans 1.17 −4 +++ + +++ +++ + 1.170 +++ + ++ +++ + 1.17 3 + + + +++ + 1.17 5 + + + +++ + Exitus d15 1.18−6 ++ (+) + +++ − C. parapsilosis 1.18 2 ++ + + ++ − 1.18 6 + +++ ++++++ − 1.18 13 + +++ − + + Exitus d13 1.19 −3 − − − + − C. albicans 1.190 − + − + − 1.19 4 − + − + − 1.19 7 + + − ++ − Exitus d7 1.22−11 + + + + + C. albicans 1.22 −7 + + − + + 1.22 0 + + + ++ + 1.222 + + + + + 1.22 7 + + + + + Discharge d7 1.25 −2 − + − ++ − C. albicans1.25 1 + − + +++ − 1.25 4 + − − ++ − 1.25 14 + (+) − ++ − 1.25 26 + + −++ − 1.26 −9 − + (+) ++ − C. parapsilosis 1.26 −5 + + + +++ − 1.260 + + + +++ − 1.26 9 + ++ + +++ − 1.26 16 + + + ++ − 1.30 −25 + (+) ? +?C. albicans 1.30 −4 + + +++ ++++ 1.30 0 + − +++ ++++ 1.30 3 + − ? +?1.30 56 + + ? −? Exitus d70 1.31 −4 ++ − ++ +? C. albicans 1.31 −1 + +++ ++++ 1.31 6 ++ (+) ++ ++++ 1.31 13 ++ − ++ ++++ Exitus d32 1.32 −19 −− + − C. parapsilosis 1.32 −17 − + + − 1.32 0 − − + − 1.32 7 − + + −1.32 15 − + + − Discharge d33 Scoring: Applicant Platelia Platelia Ab AgAb Spanish B-Ab Spanish GT Ab 0-10 = − <0.5 = − <1 = − <20 = − <20 = −10-20 = + 0.5-5 = + 1-10 = + 20-80 = + 20-200 = + 20-30 = ++ 5-10 = ++10-20 = ++ 80-600 = ++ 200-600 = ++ >30 = +++ >10 = +++ >20 = +++600-5000 = +++ 600-1200 = +++ >5000 = ++++ >1200 = ++++

TABLE 4 Patients With Negative Blood Culture Day Applicant PlateliaPlatelia Patient extraction Triple Ag Ag Ab Spanish Anti-B SpanishAnti-GT 2.2 1 ++ + − 2.2 5 ++ + − 2.2 6 ++ + (+) 2.4 1 + (+) ND 2.4 15++ + ND 2.4 17 + + ND 2.7 1 + − − + − 2.7 4 ++ + + +++ − 2.7 8 + + + +++− 2.7 11 + + + +++ − 2.7 15 + + + +++ − 2.10 1 + − +++ − 2.10 3 ++ − +++− 2.10 7 ++ − +++ − 2.10 11 ++ − ++++ − 2.10 15 ++ − +++ − 2.14 1 ++(+) + +++ − 2.14 3 ++ − + +++ − 2.14 6 ++ − + +++ − 2.14 9 ++ (+) + +++− 2.14 12 ++ + + +++ − 2.18 1 − − − 2.18 4 + + − 2.18 8 + − − 2.1812 + + − 2.18 22 + + + 2.26 1 + − − 2.26 9 + + + 2.26 16 + − + 2.2623 + + + 2.26 30 + + + 2.49 1 − − − 2.49 11 + − − 2.49 15 − − − 2.49 18− − (+) 2.49 27 + − + 2.50 1 + (+) + 2.50 9 + + (+) 2.50 15 + − + 2.5022 + − + 2.50 26 + − + 2.51 1 − − − 2.51 11 − − (+) 2.51 18 − + − 2.5122 − + − 2.51 29 + − − 2.52 1 + − (+) 2.52 8 + − − 2.52 11 + − (+) 2.5215 + − (+) 2.52 18 + − (+) 2.53 1 + − − 2.53 8 + − − 2.53 11 + + (+)2.53 18 + − − 2.53 22 + − − Scoring: Rockeby Ab Platelia Ag Platelia AbSpanish B-Ab Spanish GT Ab 0-10 = − <0.5 = − <1 = − <20 = − <20 = −10-20 = + 0.5-5 = + 1-10 = + 20-80 = + 20-200 = + 20-30 = ++ 5-10 = ++10-20 = ++ 80-600 = ++ 200-600 = ++ >30 = +++ >10 = +++ >20 = +++600-5000 = +++ 600-1200 = +++ >5000 = ++++ >1200 = ++++

TABLE 5 New Patients and Blood Donor Controls Day Blood ApplicantPlatelia Platelia Spanish Spanish Patient extraction Culture RESULT AgAb Anti-B Anti-GT B. Donor 1 N/A − − ND ND ND ND B. Donor 2 N/A − − NDND ND ND B. Donor 3 N/A − − ND ND ND ND New Patients 1 N/A − − ND ND NDND 2 N/A − (+) ND ND ND ND 3 N/A Asperg + ND ND ND ND 4 ? C. g + ND NDND ND 4 ? C. g +++ ND ND ND ND 5 ? C. a ++ ND ND ND ND 5 ? C. a +++ NDND ND ND Legend: Culture Species Applicant Ab C. a = C. albicans 0-10 =− C. g = C. glabrata 10-20 = + 20-30 = ++ >30 = +++

Table 6 summarises the original Spanish data split into the two groupsof patients, one blood culture positive and the other blood culturenegative. Of the blood culture positive group, the Applicant antigentest identified 8/11 patients as positive before they became bloodculture positive. Ultimately 10/11 patients were positive with theApplicant antigen test. Only one patient (1.32) remained negative. Thispatient was also negative by the Spanish germ tube antibody test and wasonly transiently positive by the Platelia (BioRad) mannan antigen test.It may be possible that this patient had a transient candidemia.

TABLE 6 Summary of Spanish Data Pla- Pla- Applicant telia telia SpanishSpanish Triple Ag Ag Ab Anti-B Anti-GT Culture Positive PatientsNegative result 1 0 1 0 4 Positive before culture 8 11 9 8 6 Positiveafter culture 2 1 0 1 Total Patients tested 11 11 11 8 11 CultureNegative Patients Negative result 0 3 0 0 6 Positive result 12 9 2 3 5Total Patients tested 12 12 2 3 11 Patient ID 1.01 pos pos pos pos pos1.11 pos pos pos pos pos 1.17 pos pos pos pos pos 1.18 pos pos pos pos(pos) 1.19 (pos) pos neg pos neg 1.22 pos pos pos pos pos 1.25 pos pos(pos) pos neg 1.26 pos pos pos pos neg 1.30 pos pos pos ND pos 1.31 pospos pos ND pos 1.32 neg pos pos ND neg 2.02 pos pos neg 2.04 pos pos2.07 pos pos pos pos neg 2.10 pos neg pos neg 2.14 pos (pos) pos pos neg2.18 pos pos (pos) 2.26 pos pos pos 2.49 pos neg (pos) 2.50 pos (pos)pos 2.51 (pos) pos neg 2.52 pos neg neg 2.53 pos (pos) neg

The Applicant antigen test identified all 12 of the blood culturenegative patient group as being positive for Candida antibody. Incomparison, the Platelia Mannan antigen test identified 9/12 patients asbeing positive compared to 5/11 by the Spanish Germ tube antibody test.The main problem with the blood culture negative patient group was thatthere was no other confirmation of diagnosis.

Overall, there was good correlation with the results of the Applicantantigen test and that of the other serology tests used by the Spanishgroup. Where a patient was strongly positive by the Applicant antigentest (ie.: patient's 1.11, 1.17, 1.18 and 1.31), they were also stronglypositive by the other tests. Also, where sera were negative or lowpositive by the Applicant antigen test, they are also usually negativeor weakly positive by the other tests. For example, patients 1.19, 1.22,1.25, 1.26, 1.32, 2.18, 2.26, 2.49, 2.50, 2.51, 2.52 and 2.53.

Where fresh sera was analysed (Table 5), there was a perfect correlationwith the Applicant antigen test and whether the sera was blood culturepositive or negative.

The statistical analysis of these data is presented in FIG. 9 and Table7. From the error plot diagram in FIG. 9, it is evident that the groupof patients with positive blood culture have a higher Candida antibodylevels detected by Syscan3 (mean 25.86, 95% CI: 16.28-35.44) as comparedto the patients with negative blood culture as a group (mean 17.30, 95%CI: 13.42-21.19). Comparing the means using the Independent SamplesT-Test, the difference between the two groups is statisticallysignificant at p=0.087.

TABLE 7 Mean and 95% Confidence Interval of Mean of Applicant AntigenTest Scores 95% Confidence Category Mean (Units) Interval Positive Blood25.86* 16.28-35.44 Culture Negative Blood 17.30* 13.42-21.19 Culture *p= 0.087

Example 5 Clinical Evaluation in Australia

Sera collected from patients with invasive candidiasis was obtained froman Australian hospital (1997 to 1998), the patients had haematologicalmalignancies (n=24). Control sera were collected from males 18 to 25years of age (n=20) with no history of Candida infection. The patientsera were tested with the Applicant antigen test as described in Example2. Each sera was tested in triplicate and the average reading used. Theaverage absorbance reading for each serum was divided by that of the“cut-off” calibrator serum supplied with the Applicant antigen test.This value was then multiplied by 10 to give a value in arbitrary units.

The results of the Applicant antigen test using a value of 20 units (twotimes the cut-off calibrator serum value) or above as defining apositive sample is presented in Table 8.

TABLE 8 Results of the Applicant antigen test using 20 units as acut-off Invasive Healthy Candidiasis Controls Total Test Positive 20 020 Test Negative 4 20 24 Total 24 20 44

With the Applicant antigen test using 20 units as cut-off, thespecificity of the test was 100% and sensitivity was 83.3%. —Positivepredictive value was 83.3% and negative predictive value was 100%. Whenthe value of the test considered positive was set at 10 units or 1 timesthe value of the cut-off sera absorbance, the specificity of the testdecreased, but the sensitivity increased (Table 9). The specificity was90%, sensitivity 87.5%. Positive predictive factor increased to 91.3%,while negative predictive factor decreased to 85.7%.

TABLE 9 Results of the Applicant antigen test using 10 units as acut-off Invasive Healthy Candidiasis Controls Total Test Positive 21 223 Test Negative 3 18 21 Total 24 20 44

The results of the Applicant antigen test using sera from patients withinvasive candidiasis are presented in Table 10. Only one of the negativeresults came from a patient who was blood culture positive. Two of thefour negative samples were from patients with central linecontaminations. Three of the four negative test results came frompatients with Candida parapsilosis infections, the other being C.albicans.

TABLE 10 Sera from Patients with Invasive Candidiasis Test withApplicant Antigen Test Abs Patient (420 nm) Units Result Site ofisolation Candida spp. A 1.69 61 Positive blood culture parapsilosis B0.15 5 Negative blood culture parapsilosis C 1.16 42 Positive peritonealguillermondii cavity D 1.85 67 Positive blood culture albicans E 1.16 42Positive blood culture albicans F 1.49 54 Positive peritoneal glabratacavity G 0.56 20 Positive peritoneal parapsilosis cavity I 1.24 45Positive cathater albicans K 0.98 35 Positive oesophagus albicans L 3.46124 Positive peritoneal albicans cavity M 0.19 7 Negative central linealbicans N 1.44 52 Positive wound albicans P 0.55 20 Positive sputumglabrata Q 1.12 40 Positive sputum tropicalis R 1.02 37 Positive centralline albicans S 0.88 32 Positive blood culture albicans T 1.6 58Positive blood culture glabrata U 0.22 8 Negative central lineparapsilosis V 0.55 20 Positive urine parapsilosis W 0.31 11 Negativeperitoneal parapsilosis cavity X 0.59 21 Positive central line albicansY 0.85 31 Positive blood culture albicans Z 1.06 38 Positive bronchalbicans ZA 1.05 38 Positive urine tropicalis

The Applicant antigen test data for the invasive candidiasis group andhealthy controls are presented in the error bar diagram as seen in FIG.10 and Table 11. In FIG. 10, the group with invasive candidiasis has ahigher mean (31.45 units) as compared to the healthy blood donor group(7.52 units). This difference was statistically significant (p<0.01).The 95% confidence interval range of the means was higher as well in theinvasive candidiasis group (23.57-39.33 units), as compared to thehealthy donor group (6.92-8.12 units).

TABLE 11 Mean and 95% Confidence Interval of the Mean of Patients withInvasive Candidiasis and healthy blood donors 95% CI of Group Mean(Units) mean (Units) Invasive 31.45 23.57-39.33 Candidiasis Healthyblood 7.52 6.92-8.12 donors

In this study the Applicant antigen test was used to test sera frompatients with invasive candidiasis, superficial candidiasis (oral orvaginal thrush) and healthy male controls. As a commensal organism,healthy individuals can have a measurable antibody titre to Candidaantigens. In order to differentiate between normal and infectionassociated antibody levels a cut-off calibrator serum was supplied. Theabsorbance of the serum being tested was divided by the cut-offcalibrator serum absorbance and multiplied by 10 to give an arbitraryunit value. Using a value of 20 units or above as an indicator of apositive test gave the greatest discrimination between the patient groupwith invasive candidiasis and the healthy controls (positive predictivevalue of 83%, negative predictive value 100%). If the value at which asample was considered positive was lowered to 10 units (ie. the cut-offcalibrator value), the positive predictive value increased slightly to87.5% but the negative predictive value decreased to 90%.

Only one patient with a positive blood culture returned a negative testresult with the Applicant antigen test. Two out of the four negativesera were from patients with a central line contamination. This couldtherefore reflect a transient infection in these patients, which may notprovoke an antibody response. It is of interest that 3 of the 4 negativetests were due to C. parapsilosis infections. This organism isfrequently associated with biofilms, which may shield it from the hostimmune response.

In conclusion, the Applicant antigen test is a rapid, reliable and easytest to perform. It showed good sensitivity and specificity in thediagnosis of invasive and severe superficial Candida infections.

Example 6 Comparative Test of Cytoplasmic Antigen of Present ApplicationAgainst Antigens Disclosed in U.S. Pat. No. 4,806,465

U.S. Pat. No. 4,806,465 in the name of Buckley et al. disclosesmannan-depleted cytoplasmic extract of mycelium, fractionated by ionexchange chromatography. In contrast, as described in the Examplesabove, the cytoplasmic antigens of the present invention are isolatedfrom blastospore. Moreover, the antigens isolated in the presentinvention had molecular weights of 55 kDa, 30 kDa and 20 kDa. Incontrast, the molecular weights of the antigens isolated by Buckley etal. are 120 to 135 Kd, 48 to 52 Kd and 35 to 38 Kd.

Notwithstanding, we decided to test the antigens prepared in the presentinvention with the monoclonal antibodies described in Buckley et al.that specifically bound the antigens prepared by Buckley et al.

Samples of the hybridoma cell lines ATCC #HB-8397 and ATCC #HB-8398,were obtained. These hybridomas produce monoclonal antibodies that aremonospecific to the cytoplasmic antigens disclosed in U.S. Pat. No.4,806,465 (see abstract of U.S. Pat. No. 4,806,465). These monoclonalantibodies were tested against samples of the antigens disclosed in theExamples above and briefly, none of these reacted with any of theantigens disclosed. FIG. 11, shows the results of the experiments. PanelA shows the immunoblot of C. albicans cytoplasmic (enolase) antigendisclosed in Buckley et al., while panel B shows the C. albicans antigenas disclosed in the present application.

1. A method for monitoring the treatment of a subject with a Candida infection comprising the steps of: (a) determining levels of antibody to a soluble cytoplasmic antigen preparation consisting essentially of Candida antigens of molecular weights 55 kDa, 30 kDa and 20 kDa in a subject infected with Candida; (b) administering a pharmaceutical composition for treating Candida infection to the subject; and (c) re-determining said levels of antibody, wherein said levels of antibody decrease over time if the treatment is successful.
 2. The method of claim 1, wherein the antigen preparation further comprises one or more antigens selected from the group consisting of cell wall and enolase antigen.
 3. The method according to claim 1, wherein step of determining levels of antibody comprises the steps of: a). obtaining a biological sample from a subject with a Candida infection; b). contacting said biological sample with said soluble cytoplasmic antigen preparation; and c). using a detection system to determine levels of bound antibody.
 4. The method according to claim 3, wherein the detection system is selected from the group consisting of enzyme-linked immunoassay, biligand binding, fluorometric assay, chemiluminescent assay, radialimmunodiffusion and radioimmunoassay.
 5. The method of claim 3, wherein the immunoassay is ELISA.
 6. A method according to claim 1, wherein the soluble cytoplasmic antigen preparation is bound to a solid phase either by adsorptive binding, covalent binding, or via a ligand already bound to the solid phase.
 7. A method according to claim 3, further comprising the step of using secondary labelled antibodies to detect the antibodies to Candida present in the biological samples.
 8. A method according to claim 7, further comprising the step of labelling the secondary antibodies with a label selected from the group consisting of fluorescent dye, radioisotope, enzyme, or combinations thereof.
 9. A method according to claim 7, wherein the secondary antibody is labelled via bound ligands.
 10. A method according to claim 3, wherein detection in the detection system is selected from the group consisting of colour development, chemiluminescence, fluorescence, radioactivity, or combinations thereof.
 11. A method according to claim 3, further comprising the step of performing the detection of antibodies by a method selected from the group consisting of qualitative detection, quantitative detection, or combination thereof.
 12. A method according to claim 8, further comprising the step of directly labelling the secondary antibody.
 13. A method according to claim 8, further comprising the step of indirectly labelling the secondary antibody.
 14. A method according to claim 3, wherein the antigen composition is either immobilized on an inert surface, embedded in a gel, or conjugated to a molecule.
 15. A method according to claim 14, wherein the molecule imparts colour, fluorescence or radioactivity to the antigen.
 16. A method according to claim 3, wherein the biological sample is selected from the group consisting of bone marrow, plasma, spinal fluid, lymph fluid, skin, tears, saliva, milk, blood, serum, blood cells, tumors and organs.
 17. A method according to claim 16, wherein the skin consists of external sections selected from the group consisting of respiratory, intestinal, and genitourinary tracts.
 18. A method according to claim 17, wherein the biological sample is serum.
 19. A lateral flow device for the detection of Candida antibody in a biological sample comprising a membrane having a detection zone and a control zone, and a wicking pad wherein said detection zone includes a soluble cytoplasmic antigen preparation consisting essentially of Candida antigens of molecular weights 55 kDa, 30 kDa and 20 kDa.
 20. The lateral flow device of claim 19, further comprising a sample pad.
 21. The lateral flow device of claim 19, wherein the biological sample deposited on an end of said detection zone is wicked into said detection zone by capillary action.
 22. A method for detecting the presence or absence of Candida antibodies comprising the step collecting a biological sample from a subject at risk of, or suspected to be suffering from, Candida infection and applying said sample onto a lateral flow device of claim
 19. 23. The method of claim 3, wherein in the immunoassay is an immunoblot.
 24. The method of claim 3, wherein the immunoassay is a protein array. 